The present invention relates to active health promoting ingredients from red grape cells (RGC), more particularly, to therapeutic formulations thereof.
Modern scientific thought is that chronic heart disease (CHD) and strokes are symptoms of a common illness related to a lack of cardiovascular (CV) health. Worldwide CV diseases account for half of all deaths in middle age (and considerable additional disability) and one third of all deaths in old age. Most of these deaths involve ischemic heart disease (IHD) or stroke (Lewington, 2003, Eur. Heart J. 24:1703-1704). It also incurs a substantial burden on health budgets primarily due to the high cost of hospitalization and ambulatory medical management. Although heart attacks are considered to be the major cause of death in men, recent studies have clearly shown that heart attack will strike one out of three women as well.
The process of coronary atherosclerosis development, leading to acute coronary syndromes, is comprised of four subsequent patho-physiological stages: endothelial dysfunction, plaque formation, plaque development and plaque rupture and thrombosis.
Endothelial dysfunction is the disruption of the functional integrity of the vascular endothelium as a result of exposure to cardiovascular risk factors (such as elevated LDL levels, smoking, hypertension etc.). Dysfunctional endothelium devoid of its protective properties allows the action of atherogenetic factors on the vessel wall and promotes inflammation within the wall, thus leading to increased monocyte activation, adhesion and migration, increased endothelial permeability and reduced vasodilation, thereby mediating accumulation of macrophages and lipoproteins within the wall.
Atherosclerotic plaques are formed when macrophages ingest chemically modified (usually oxidized) LDL molecules to form foam cells, which together with the T-cells and vascular smooth muscle cells (VSMC) create fatty streaks, the early form of the atherosclerotic plaque.
Inflammatory mediators and other molecules promote further development of the plaque into a fibro-fatty atheroma, which later becomes covered with a fibrous cap with a dense extracellular matrix. This cap stabilizes the plaque from rupture by making it larger.
Secretion of molecules (e.g. inflammatory molecules) by the foam cells leads to digestion of the cap matrix molecules, ultimately leading to plaque rupture, formation of a thrombus (clot) and arterial occlusion. This typically leads to a heart attack or a stroke.
Until a few years ago, most physicians considered atherosclerosis as a plumbing problem, caused by a halting of arterial blood flow by a plaque which had reached a particular size. It was claimed that the shortage of oxygenated blood to indispensable tissues, such as the cardiac muscle or brain tissue, especially at critical moments of greater need, was responsible for the induced heart or brain stroke. However, recent studies have clearly shown that in fact only about 15% of heart attacks happen this way. Pathological and other studies have demonstrated that events, which follow inflammatory processes leading to breakage of the plaque fibrous cap and resulting in blood clotting, are responsible for most heart attacks and brain strokes [Libby, 2002, Nature 420:868-874; Libby, 2004, Sci. Am. Special edition 14:50-59]. Since inflammatory processes are involved in all steps of atherogenesis, from endothelial dysfunction to plaque rupture, interference with these inflammatory mechanisms may help to prevent or fight atherosclerosis.
This new view of atherosclerosis explains the limited success and unwanted side effects of some of the medical treatments of atherosclerosis, developed during the last twenty years. For example, balloon angioplasty and stents may mediate rupturing of the residual plaques, thus eliciting strong inflammatory response. The present strategy of medically treating atherosclerosis emphasizes prevention of plaque creation and development of drugs that may cope with the processes leading to inflammation and clot formation. Such current in-use drugs include statins (inhibition of LDL biosynthesis); beta-blockers (reduce hypertension or pulse rate); aspirin (helps in prevention of inflammation or blood clotting); and anti-oxidants (prevention of LDL modification).
A strict correlation between reduction of deaths from heart diseases and increased wine consumption was reported twenty five years ago. Substantial studies clearly demonstrated the positive effect, unrelated to alcohol, of moderate red-wine consumption on coronary heart disease (CHD) mortality, known as the “French Paradox” [Renaud and de Lorgeril, 1992, Lancet 339:1523-1526; Criqui and Ringel, 1994, Lancet 344:1719-1723]. Moreover, it was suggested that polyphenols, which are present at higher concentration in red rather than white wine, act as antioxidants that protect blood low-density lipoproteins (LDL) from oxidation, a modification that is known to be a key risk factor in the development of CHD.
Recent results have demonstrated the participation of several proteins in the inflammatory processes which leads to CHD, whose levels may be regulated by constituents present in red wine: Endothelin-1 (ET-1), a potent vasoactive peptide (Kinlay et al. 2001, Curr. Opin. Lipidol. 12:383-389); endothelial nitric oxide synthase (eNOS), NO producer in endothelial cells (Leikert et al. 2002, Circulation 106:1614-1617); the platelet-derived growth factor (PDGF) which is active in VSMC (Iijima et al. 2002, Circulation 105:2404-2410) and the inflammatory marker C-reactive Protein (CRP; Aikawa & Libby, 2004, Can. J. Cardiol. 20:631-634).
Use of red wine as a source of these regulatory constituents is limited due to its high alcoholic content. Likewise, use of grapes or grape juice as a source of these active agents is limited due to their high sugar content. In addition, it has been shown that the therapeutic effect of wine and wine grapes is dependant on species, location, year (annual climate), processing etc. and therefore reliance on red wine or edible grapes as a source for consumption of these regulatory compounds does not lead to a homogeneous or consistent supply of material. Furthermore, grapes are typically contaminated by residual fungicides, pathogens, pesticides and pollutants.
A major problem associated with the potential benefit of polyphenols present in red wines and grape seed extracts lies in their bioavailability to target tissues and cells (Manach and Donovan, 2004, Free Rad. Res. 38:771-785; Williamson & Manach, 2005, Am. J. Clin. Nutr. 81:243 S-255S). Due to marked differences in their bioavailability while passing through the intestines, no obvious correlation can be drawn between the abundance of a certain polyphenol in a given food and its concentration as an active compound in vivo. The absorbance of flavonoids in the small intestines, for example, ranges from 0-60% of the dose, and elimination half-lives range from 2-48 hours [Manach and Donovan, 2004, supra]. Most polyphenols undergo extensive metabolism in the intestine, and are present in serum and urine predominantly as glucuronides, methyl or sulfate conjugates.
The mucous layer in the mouth is the potential site that provides improved absorption rates for the beneficial polyphenols. The bioavailability of trans-Resveratrol was reported to be increased if, instead of being immediately swallowed, the polyphenols were retained in the mouth for one minute before swallowing; considerable amounts of trans-Resveratrol were then measured in the plasma just two minutes after administration (Asensi et al, 2002, Free Radic Biol Med. 33:387-398). In addition, recent epidemiologic evidence supports the view that dietary flavonoids exert protective effects in oral diseases, including cancer (Browning et al, 2005, J Pharm Pharmacol. 57:1037-42) if activated by the saliva.
PCT patent application publication WO 00/35298 and PCT patent application publication WO 01/21156 teach chewing gum containing medicament active agents, including nutritional supplements such as grape seed extracts and polyphenols. Fruit cell culture preparations (e.g., extracts), or fruit cell line culture extracts are not disclosed. European patent application EP 1327441 teaches a chewing gum composition useful for reducing nicotine exposure in a subject. The agent that eliminates nicotine is an extract of a mixture of different plants, fruit and the polyphenols quercitin or catechin. Grape skin extracts are mentioned as natural pigments to color the chewing gum and not as active agents. As mentioned herein above, both grape skin and grape seed extracts provide non-defined, non-consistent and non-homogenous active agents. Unlike skin or seed extracts, fruit cell culture, and more particularly fruit cell line culture, produces highly defined phytochemicals without many of the associated interfering compounds such as sugars and pectins, in a tightly controlled environment that can be manipulated to influence the types and amounts of active compounds.
There is thus a widely recognized need for, and it would be highly advantageous to have a fruit cell, and more particularly a grape cell line extract, rich in active agents formulated to enhance bioavailability for the treatment of inflammatory disorders.